Immune checkpoint inhibitor therapies targeting PD-1/PD-L1 are now the standard of care in oncology across several hematologic and solid tumor types, including triple negative breast cancer (TNBC). Patients with metastatic or locally advanced TNBC with PD-L1 expression on immune cells occupying ≥1% of tumor area demonstrated survival benefit with the addition of atezolizumab to nab-paclitaxel. However, concerns regarding variability between immunohistochemical PD-L1 assay performance and inter-reader reproducibility have been raised. High tumor-infiltrating lymphocytes (TILs) have also been associated with response to PD-1/PD-L1 inhibitors in patients with breast cancer (BC). TILs can be easily assessed on hematoxylin and eosin-stained slides and have shown reliable inter-reader reproducibility. As an established prognostic factor in early stage TNBC, TILs are soon anticipated to be reported in daily practice in many pathology laboratories worldwide. Because TILs and PD-L1 are parts of an immunological spectrum in BC, we propose the systematic implementation of combined PD-L1 and TIL analyses as a more comprehensive immuno-oncological biomarker for patient selection for PD-1/PD-L1 inhibition-based therapy in patients with BC. Although practical and regulatory considerations differ by jurisdiction, the pathology community has the responsibility to patients to implement assays that lead to optimal patient selection. We propose herewith a riskmanagement framework that may help mitigate the risks of suboptimal patient selection for immuno-therapeutic approaches in clinical trials and daily practice based on combined TILs/PD-L1 assessment in BC.

Stromal tumor-infiltrating lymphocytes (sTILs) are important prognostic and predictive biomarkers in triple-negative (TNBC) and HER2-positive breast cancer. Incorporating sTILs into clinical practice necessitates reproducible assessment. Previously developed standardized scoring guidelines have been widely embraced by the clinical and research communities. We evaluated sources of variability in sTIL assessment by pathologists in three previous sTIL ring studies. We identify common challenges and evaluate impact of discrepancies on outcome estimates in early TNBC using a newly-developed prognostic tool. Discordant sTIL assessment is driven by heterogeneity in lymphocyte distribution. Additional factors include: technical slide-related issues; scoring outside the tumor boundary; tumors with minimal assessable stroma; including lymphocytes associated with other structures; and including other inflammatory cells. Small variations in sTIL assessment modestly alter risk estimation in early TNBC but have the potential to affect treatment selection if cutpoints are employed. Scoring and averaging multiple areas, as well as use of reference images, improve consistency of sTIL evaluation. Moreover, to assist in avoiding the pitfalls identified in this analysis, we developed an educational resource available at www.tilsinbreastcancer.org/pitfalls.

The advent of immune-checkpoint inhibitors (ICI) in modern oncology has significantly improved survival in several cancer settings. A subgroup of women with breast cancer (BC) has immunogenic infiltration of lymphocytes with expression of programmed death-ligand 1 (PD-L1). These patients may potentially benefit from ICI targeting the programmed death 1 (PD-1)/PD-L1 signaling axis. The use of tumor-infiltrating lymphocytes (TILs) as predictive and prognostic biomarkers has been under intense examination. Emerging data suggest that TILs are associated with response to both cytotoxic treatments and immunotherapy, particularly for patients with triple-negative BC. In this review from The International Immuno-Oncology Biomarker Working Group, we discuss (a) the biological understanding of TILs, (b) their analytical and clinical validity and efforts toward the clinical utility in BC, and (c) the current status of PD-L1 and TIL testing across different continents, including experiences from low-to-middle-income countries, incorporating also the view of a patient advocate. This information will help set the stage for future approaches to optimize the understanding and clinical utilization of TIL analysis in patients with BC.

Pilocytic astrocytomas (PAs) are increasingly tested for KIAA1549-BRAF fusions. We used reverse transcription polymerase chain reaction for the 3 most common KIAA1549-BRAF fusions, together with BRAF V600E and histone H3.3 K27M analyses to identify relationships of these molecular characteristics with clinical features in a cohort of 32 PA patients. In this group, the overall BRAF fusion detection rate was 24 (75%). Ten (42%) of the 24 had the 16-9 fusion, 8 (33%) had only the 15-9 fusion, and 1 (4%) of the patients had only the 16-11 fusion. In the PAs with only the 15-9 fusion, 1 PA was in the cerebellum and 7 were centered in the midline outside of the cerebellum, that is, in the hypothalamus (n = 4), optic pathways (n = 2), and brainstem (n = 1). Tumors within the cerebellum were negatively associated with fusion 15-9. Seven (22%) of the 32 patients had tumor-related deaths and 25 of the patients (78%) were alive between 2 and 14 years after initial biopsy. Age, sex, tumor location, 16-9 fusion, and 15-9 fusion were not associated with overall survival. Thus, in this small cohort, 15-9 KIAA1549-BRAF fusion was associated with midline PAs located outside of the cerebellum; these tumors, which are generally difficult to resect, are prone to recurrence.

Background The pan-histone deacetylase inhibitor panobinostat is a potential therapy for malignant glioma, but it is water insoluble and does not cross the blood–brain barrier when administered systemically. In this article, we describe the in vitro and in vivo efficacy of a novel water-soluble nano-micellar formulation of panobinostat designed for administration by convection enhanced delivery (CED). Materials and methods The in vitro efficacy of panobinostat-loaded nano-micelles against rat F98, human U87-MG and M059K glioma cells and against patient-derived glioma stem cells was measured using a cell viability assay. Nano-micelle distribution in rat brain was analyzed following acute CED using rhodamine-labeled nano-micelles, and toxicity was assayed using immunofluorescent microscopy and synaptophysin enzyme-linked immunosorbent assay. We compared the survival of the bioluminescent syngenic F98/Fischer344 rat glioblastoma model treated by acute CED of panobinostat-loaded nano-micelles with that of untreated and vehicle-only-treated controls. Results Nano-micellar panobinostat is cytotoxic to rat and human glioma cells in vitro in a dose-dependent manner following short-time exposure to drug. Fluorescent rhodamine-labelled nano-micelles distribute with a volume of infusion/volume of distribution (Vi/Vd) ratio of four and five respectively after administration by CED. Administration was not associated with any toxicity when compared to controls. CED of panobinostat-loaded nano-micelles was associated with significantly improved survival when compared to controls (n=8 per group; log-rank test, P <0.001). One hundred percent of treated animals survived the 60-day experimental period and had tumour response on post-mortem histological examination. Conclusion CED of nano-micellar panobinostat represents a potential novel therapeutic option for malignant glioma and warrants translation into the clinic.

Introduction. EGFR and EGFRvIII analysis is of current interest because of new EGFRvIII vaccine trials opened in the UK. EGFR activation promotes cellular proliferation via activation of MAPK and PI3K-Akt pathways. EGFRvIII is the most common variant resulting from an in-frame deletion of 801bp, leading to constitutively active EGFR. Method. 51 glioblastoma samples from a cohort of 50 patients were tested for EGFR amplification by FISH and immunohistochemistry and EGFRvIII expression by reverse-transcriptase PCR (RT-PCR), and immunohistochemistry. EGFR and EGFRvIII expression was compared with Overall Survival in the cohort. Results. Overall 22/51 samples (43%) were positive for EGFR, 16/51 (31%) were positive for EGFRvIII and 13/51 (25%) were positive for both. 9/51 cases (18%) were positive for EGFR alone, and 3/51 (6%) were positive for EGFRvIII alone. Of the EGFR positive cases, 22/51 (43%) were positive by FISH, 24/51 (47%) were positive by IHC and 2/51 (4%) were discrepant between methods (positive by IHC but non-amplified by FISH). Of the EGFRvIII positive cases, 16/51 (31%) were positive by RT-PCR, 17/51 (33%) were positive by IHC and 1/51 (2%) sample was discrepant (positive by IHC but not by RT-PCR). Neither EGFRvIII or EGFR are predictive of overall survival in this cohort. Conclusion. In our cohort, 25/51 (49%) of GBM showed EGFR alterations, including 16/51 (31%) with EGFRvIII. There was high concordance between IHC and FISH (96%) and IHC and RT-PCR (98%) as diagnostic methods. Neither EGFR or EGFRvIII is predictive of overall survival in this cohort. These results are key for selecting patients for novel individualised anti-EGFR therapies.

ObjectivesFriedreich's ataxia is a devastating neurological disease currently lacking any proven treatment. We studied the neuroprotective effects of the cytokines, granulocyte‐colony stimulating factor (G‐CSF) and stem cell factor (SCF) in a humanized murine model of Friedreich's ataxia.MethodsMice received monthly subcutaneous infusions of cytokines while also being assessed at monthly time points using an extensive range of behavioral motor performance tests. After 6 months of treatment, neurophysiological evaluation of both sensory and motor nerve conduction was performed. Subsequently, mice were sacrificed for messenger RNA, protein, and histological analysis of the dorsal root ganglia, spinal cord, and cerebellum.ResultsCytokine administration resulted in significant reversal of biochemical, neuropathological, neurophysiological, and behavioural deficits associated with Friedreich's ataxia. Both G‐CSF and SCF had pronounced effects on frataxin levels (the primary molecular defect in the pathogenesis of the disease) and a regulators of frataxin expression. Sustained improvements in motor coordination and locomotor activity were observed, even after onset of neurological symptoms. Treatment also restored the duration of sensory nerve compound potentials. Improvements in peripheral nerve conduction positively correlated with cytokine‐induced increases in frataxin expression, providing a link between increases in frataxin and neurophysiological function. Abrogation of disease‐related pathology was also evident, with reductions in inflammation/gliosis and increased neural stem cell numbers in areas of tissue injury.InterpretationThese experiments show that cytokines already clinically used in other conditions offer the prospect of a novel, rapidly translatable, disease‐modifying, and neuroprotective treatment for Friedreich's ataxia. Ann Neurol 2017;81:212–226

It is increasingly clear that both adult and pediatric glial tumor entities represent collections of neoplastic lesions, each with individual pathological molecular events and treatment responses. In this review, we discuss the current prognostic biomarkers validated for clinical use or with future clinical validity for gliomas. Accurate prognostication is crucial for managing patients as treatments may be associated with high morbidity and the benefits of high risk interventions must be judged by the treating clinicians. We also review biomarkers with predictive validity, which may become clinically relevant with the development of targeted therapies for adult and pediatric gliomas.